Antibody-based systems for diagnosis and therapy rest on development of chemistry for the covalent modification of antibodies, especially, monoclonal antibodies, as well as the further development of linker systems which can, for example, enhance the performance of drugs delivered by the antibody.
An important factor in the use of antibody-based systems concerns the quality and nature of the antibody conjugate used. Originally, aggregated antibody enzyme conjugates were produced using a nonspecific coupling chemistry such as glutaraldehyde crosslinking which was described by Avrameas, Immunochemistry, 6:43 (1969) or a periodate oxidation method as described by Boorsma et al., Histochem. Cytochem., 23:200 (1974). The drawback with these approaches is that crosslinking is random and the antibodies are often buried deep within the resulting aggregated complex. Accordingly, the relative inaccessibility of antibodies reduces, if not eliminates altogether, their specific activity.
Recently, several approaches have been described in which both proteins, e.g., antibody and enzyme or toxin, are activated at their primary amine groups using an NHS-ester group of a heterobifunctional reagent to introduce a functional moiety such as a maleimide or sulfhydryl group. Imagawa et al., J. Appl. Biochem., 4:400 (1982), Duncan et al., Anal. Biochem., 132:68 (1983).
Three methods commonly used to thiolate involve introducing a free sulfhydryl group which is deblocked before proceeding with the conjugation. These methods include the following: (1) reaction with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)as described by Carlsson et al., Biochem. J., 173:723 (1978); or (2) reaction with 4-mercaptobutyrimidate in the presence of 4,4'-dithiodipyridine as described by King et al., Immunol. Method, 28:201 (1979); or reaction with S-acetyl mercaptosuccinic anhydride (SAMSA) as described by Weston et al., Biochem. Biophys. Acta, 612:40 (1980).
U.S. Pat. No. 4,231,999, issued to Carlsson et al. on Nov. 4, 1980, describes an assay method involving biospecific affinity reactions. Thiolation using SPDP involving subsequent reduction with dithiothreitol prior to conjugation.
U.S. Pat. No. 4,232,119, issued to Carlsson et al. on Nov. 4, 1980 describes reagents such as SPDP for use as reagents in immunoassays.
Duncan et al., Analytical Biochemistry, 132:68-73 (1983), descries the synthesis of the N-hydroxysuccinimide ester of S-acetylthioacetic acid (SATA) and its use in preparing conjugates.
Derksen et al., Biochimica at Biophysica Acta, 814:151-155 (1985), describe the use of SATA to couple proteins to liposomes containing maleimide residues.
Peeters et al., J. Immunological Methods, 120:133-143 (1989), describes a comparison of several coupling reagents such as SATA and SPDP on the antigenicity and immunogenicity of the conjugates.
The disadvantage of these approaches is that the thiolated protein tends to aggregate during the conjugation process. Protection of free sulfhydryl groups in solution from adventitious reaction, particularly oxidation, is almost impossible.